Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials in patients with hemophilia. Two AAV based gene therapy drugs have been recently approved by the FDA. Luxturna has been valued at $850,000 for a one-time treatment for a rare form of blindness and Zolgensma at $2,100,000 for spinal muscle atrophy. Gene therapy with AAV vectors has shown a potentially huge market. Although successful in clinical studies, two concerns restrict a broader AAV vector application for patients with hemophilia who need systemic administration of AAV vector for effective liver targeting: low human hepatocyte transduction and neutralizing antibody (Nab)-mediated blocking of AAV transduction. Several approaches have been explored for AAV transduction enhancement or neutralizing antibody evasion. Engineering of the AAV capsid represents a very powerful and popular technology, and has been extensively studied to develop novel AAV vectors for transduction enhancement in pre-clinical animal models or Nab escape in vitro. It has been demonstrated that the results from mouse experiments do not recapitulate those of large animals such as primates and dogs, the data for AAV variants generated in animal cells and organs may not translate into human application. Recently, a mouse model xenografted with human hepatocytes has been used to develop AAV vectors for targeting gene therapy relative to human liver. We have isolated several AAV mutants from the liver of chimeric mice xenografted with human hepatocytes in the presence of human neutralizing antibodies (IVIG) using AAV shuffling library approach. In this application, we will study the ability of these mutants to transduce human hepatocytes and evade Nabs. Following the identification of the best mutants with efficient human hepatocyte transduction and high ability for Nab evasion, we will study the safety and long-term efficacy of these mutants for hemophilia gene therapy in a mouse model with AAV Nabs after a single injection. Successful validation of these mutants in hemophilic mice will provide a proof-of-concept for translation to larger models of hemophilia in hopes of safely and effectively treating hemophilia patients with AAV Nabs using a single dose.